Claire Husser

N1‐methylpseudouridine ( ^m Ψ) is a modified nucleoside that entirely replaces uridine in the design of many therapeutic messenger RNAs (mRNA) to alleviate their intrinsic immunogenicity while preserving their capacity to be translated into protein. However, the impact of such replacement in functional non‐coding RNAs has only been scarcely studied, and most studies have shown impairment in the function of the tested RNAs thereby limiting their potential applications. In this work, we further show that exchanging uridines by ^m Ψ in green‐emitting Fluorogenic Light‐up Aptamers (FLAP) is detrimental to their capacity of binding and enhancing the fluorescence of their cognate fluorogen. Moreover, combining directed evolution and rational design allowed not only to pinpoint the structural elements impacted by the replacement of uridine by ^m Ψ, but also to re‐establish the function of a ^m Ψ‐modified non‐coding RNA for the first time. This yielded Ψ‐Spinach, a FLAP suited for the precise labelling of ^m Ψ‐modified mRNA and usable for human cells transfection. This work thus paves the way for the adaptation of other functional non‐coding RNAs with ^m Ψ in view of engineering and optimizing therapeutic modified mRNAs.

N1‐methylpseudouridine ( ^m Ψ) is a modified nucleoside that entirely replaces uridine in the design of many therapeutic messenger RNAs (mRNA) to alleviate their intrinsic immunogenicity while preserving their capacity to be translated into protein. However, the impact of such replacement in functional non‐coding RNAs has only been scarcely studied, and most studies have shown impairment in the function of the tested RNAs thereby limiting their potential applications. In this work, we further show that exchanging uridines by ^m Ψ in green‐emitting Fluorogenic Light‐up Aptamers (FLAP) is detrimental to their capacity of binding and enhancing the fluorescence of their cognate fluorogen. Moreover, combining directed evolution and rational design allowed not only to pinpoint the structural elements impacted by the replacement of uridine by ^m Ψ, but also to re‐establish the function of a ^m Ψ‐modified non‐coding RNA for the first time. This yielded Ψ‐Spinach, a FLAP suited for the precise labelling of ^m Ψ‐modified mRNA and usable for human cells transfection. This work thus paves the way for the adaptation of other functional non‐coding RNAs with ^m Ψ in view of engineering and optimizing therapeutic modified mRNAs.

Développement d’ARN synthétiques par criblage microfluidique à ultrahaut-débit: Les aptamères d'ARN sont des acides nucléiques monocaténaires capables d'adopter diverses conformations pour reconnaître spécifiquement une large gamme de ligands, y compris de petites molécules et des protéines. En plus de leur capacité de reconnaissance, ces aptamères peuvent exercer des fonctions complexes, ce qui les rend prometteurs en tant qu'outils thérapeutiques et diagnostiques. Ils trouvent des applications variées, allant de l'inhibition d'enzymes à la détection précise de petites molécules dans différents contextes. Dans cette thèse, la combinaison des méthodes SELEX et μIVC-seq a permis ’ x différentes fonctions. Elle a conduit à la sélection de biosenseurs de petites molécules avec des applications en détection environnementale, ainsi qu'au développement d'aptamères inhibiteurs d'enzymes, stables et efficaces dans des milieux complexes.

The detection of small molecules (e.g., pesticides, pollutants, antibiotics, drug residues) is critical for human health and environmental surveys as well as for many industrial applications. Fluorogenic RNA-based biosensors (FRBs) are aptamer-based tools specifically reporting on the presence of such targets by converting a specific binding event into fluorescence emission, thus making them of particular interest for on-site sensing applications. However, new sensor aptamers suited for FRB development are tedious to develop. In this study, we show that Capture-SELEX used in tandem with our microfluidic-assisted screening technology (μIVC) accelerates de novo FRB development by reprogramming the specificity of an existing sensor aptamer, followed by fluorescence-based functional selection. Furthermore, we show that these new sensor aptamers can be repurposed into functional biotechnological tools, such as gene-regulating aptazymes, thus demonstrating the versatility of our pipeline for the development of sensor aptamers adapted for various applications.

A complete genome sequence of Caballeronia sp. strain S22 capable of growing with fluoroacetate as the sole source of carbon and energy was obtained by PacBio technology. It consists of seven circular replicons totaling 9,367 kb, with a gene cluster involved in fluoroacetate utilization on its smallest 172 kb plasmid.

Nucleic acids are not only essential actors of cell life but also extremely appealing molecular objects in the development of synthetic molecules for biotechnological application, such as biosensors to report on the presence and concentration of a target ligand by emission of a measurable signal. In this work, FluorMango, a fluorogenic ribonucleic acid (RNA)‐based biosensor specific for fluoride is introduced. The molecule consists of two RNA aptamer modules, a fluoride‐specific sensor derived from the crcB riboswitch which changes its structure upon interaction with the target ion, and the light‐up RNA Mango‐III that emits fluorescence when complexed with a fluorogen. The two modules are connected by an optimized communication module identified by ultrahigh‐throughput screening, which results in extremely high fluorescence of FluorMango in the presence of fluoride, and background fluorescence in its absence. The value and efficiency of this biosensor by direct monitoring of defluorinase activity in living bacterial cells is illustrated, and the use of this new tool in future screening campaigns aiming at discovering new defluorinase activities is discussed.

RNA is instrumental to cell life in many aspects, especially gene expression regulation. Among the various known regulatory RNAs, riboswitches are particularly interesting cis‐acting molecules as they do not need cellular factor to achieve their function and are therefore highly portable from one organism to the other. These molecules usually found in the 5′ untranslated region of bacterial messenger RNAs are able to specifically sense a target ligand via an aptamer domain prior to transmitting this recognition event to an expression platform that turns on, or off, the expression of downstream genes. In addition to their obvious scientific interest, these modular molecules can also serve for the development of synthetic RNA devices with applications ranging from the control of transgene expression in gene therapy to the specific biosensing of small molecules. The engineering of such nanomachines is greatly facilitated by the proper understanding of their structure as well as the introduction of new technologies. Herein, a general overview of the current knowledge on natural riboswitches prior to explaining the main strategies used to develop new synthetic structure‐switching molecules (riboswitches or biosensors) controlled by small molecules is given.